Most of our effort on axon growth and guidance currently focuses on understanding the action of the signaling modules that translate receptor signals at the growth cone surface into guided motility of the axon tip. We focus in particular on the Abl tyrosine kinase signaling pathway, which plays a central role downstream of many of the most common, phylogenetically-conserved guidance receptors. In a completely unexpected twist, in the past year we discovered that the guidance function of Abl is executed, in part, through regulation of the secretory apparatus. Thus, under Aim 1 we have made the following discoveries: 1. The key Abl effector, the actin-regulatory protein Enabled, is selectively associated with the cis-Golgi compartment in developing Drosophila photoreceptor neurons. We have shown this by standard fluorescence microscopy, super-resolution fluorescence and by immunoelectron microscopy. 2. Loss-of function of Abl kinase, or gain-of-function of its antagonist Enabled, cause fragmentation of the Golgi apparatus, and relocalization of Golgi cisternae within the cell bodies of photoreceptor neurons. Live imaging demonstrates that reducing Abl activity increases the frequency of splitting of Golgi cisternae, and decreases the frequency of fusions. 3. Genetic and pharmacological experiments demonstrate that the effect of Abl on the Golgi structure and distribution is mediated through Abl-dependent regulation of the actin cytoskeleton. 4. Two lines of evidence suggest that the effect of Abl pathway mutations on axon guidance are mediated, in part, by their effects on secretion. First, mutations that interfere directly with Golgi function produce axonal defects that mimic those of Abl mutants. Second, genetic tests show that bona fide secretory mutants act downstream of Abl kinase in the pathway leading to axon patterning. Together, these data strongly suggest that the axonal phenotypes of Abl mutants are due, in part, to modulation of the secretory apparatus by Abl, working through the Abl-dependent regulation of actin structure and dynamics. Abl is a central regulator of axon patterning, cell polarity and epithelial organization and integrity in both vertebrates and invertebrates. It is the causative oncogene for two common forms of human leukemia, and recent evidence suggest it has a critical role in the etiology of Parkinson's disease. For three decades, studies of Abl have focused on its effects on the cortical actin cytoskeleton. Our results suggest that a vast body of work on Abl must now be reconsidered to ascertain how many of its effects are actually mediated, in part or in whole, through its effects on protein sorting, trafficking and secretion. In the past year, we have also initiated experiments under Aim 2 by developing a system for live imaging of single TSM neurons as they pioneer the L1 nerve of the developing fly wing. Prepupal wing discs from flies expressing fluorescent markers of the actin cytoskeleton or the plasma membrane in TSM are explanted and visualized by spinning disc confocal microscopy. Initial observations reveal that the morphology of pioneer neurons in situ is fundamentally different from the picture that has been derived from previous analyses of fixed samples, or from imaging axons growing in culture. The large, flat, lamellar morphology typically associated with growth cones is in fact observed only when the axon is not extending; it is the morphology associated with stationary growth cones at a choice point in their trajectory. Moreover, contrary to the common view that lamellipodia provide the motive force for growth we find that rapidly extending axons have few if any lamellipodia. Rather, axon extension arises from selective retention of filopodia. Finally, preliminary data suggest that stabilization and selective retention of leading filopodia is determined by their correlation with the local distribution of the guidance cue Delta on the underlying wing epithelium. We are currently developing metrics for quantifying cytoskeletal structures in these growth cones and analyzing their correlation with growth cone speed and direction.